Published United States patent application US2009/0075902, by certain of the inventors herein, describes peptides which block the interaction between NF-κB essential modulator (“NEMO”) with IκB kinase-β (IKK-β), the contents of which are expressly incorporated by reference.
The transcription factor NF-κB is a central component of the cellular response to damage, stress and inflammation. In mammals, the NF-κB family consists of five subunits, RelA or p65, c-Rel, RelB, p50, and p52. NF-κB binds to DNA as a dimer, the most common being the p65p50 heterodimer. The p65″p50 heterodimer is localized primarily in the cytoplasm, maintained in this inactive state via sequestration by IκB proteins. NF-κB activation via the canonical pathway is mediated by the upstream IκB kinase (IKK), a heterotrimer consisting of two catalytic subunits, IKKa and IKKß, and a regulatory subunit termed IKKγ or NEMO (NF-κB Essential Modulator). In response to a variety of factors, including pro-inflammatory cytokines, pathogens, oxidative stress and growth factors, IKK is activated and phosphorylates IκB, leading to its polyubiquitination and subsequent proteosomal degradation. IκB degradation allows NF-κB to translocate to the nucleus where it binds to its cognate DNA sequence as well as co-activators such as CBP/p300, to regulate gene expression.
Chronic activation of NF-κB is associated with numerous diseases including sepsis, asthma, muscle atrophy, multiple sclerosis, atherosclerosis, heart disease, both type I and II diabetes, osteoarthritis, dementia, osteoporosis, and cancer, most of which are associated with increasing chronologic age. NF-κB transcriptional activity is increased with age in numerous tissues in rodents including skin, liver, kidney, cerebellum, cardiac muscle, and gastric mucosa. Cells from aged humans and patients with Hutchinson-Gilford progeria have a similar upregulation of NF-κB activity. Recently, several studies suggest that NF-κB could have a causal role in aging and age-related degeneration. NF-κB was identified as the transcription factor most associated with mammalian aging. In addition, genetic inhibition of NF-κB in the skin of a transgenic mouse model reversed age-related gene expression and histologic changes, including increased epidermal thickness and reduction of senescence associated β-galactosidase activity.
An 11 amino acid sequence was identified within IKKß comprising the binding domain between IKKβ and IKKγ. This peptide, known as the NEMO binding domain (NBD), is able to block the interaction of IKKα and β (two catalytic subunits of NF-κB activation) with the regulatory subunit IKKγ (NEMO). When this short peptide sequence TALDWSWLQTE was linked to a protein transduction domain, it led to a dose dependent inhibition of NF-κB signaling in tissue culture and in animal models. While there are numerous small molecule inhibitors of NF-κB, there are some distinct advantages of the NBD peptide. The site of action is highly defined, and only activated, but the basal level of NF-κB is not affected. Also, because of the high specificity of the NBD peptide sequence it is unlikely to affect other essential kinases, which is not the case for numerous other IKK inhibitors. The NBD peptide, when attached to a protein transduction domain, has been shown to be effective in treating a wide range of inflammatory and degenerative diseases including muscular dystrophy, inflammatory bowel disease, arthritis, diabetes, accelerated aging, Parkinson's Diseases, multiple sclerosis, asthma, heart ischemia and cancer. However, the cost of synthesis of the NBD peptide fused to NBD, as well as the fact that it is not orally active limits its therapeutic application.
Certain of the inventors herein have previously disclosed 7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one and its enantiomers; BAY11-7082 or BAY11-7085 (Axxora L.L.C., San Diego, Calif.) (Petegnief et al., 2001, Neuroscience 104:223); SC-514 (Kishore et al., 2003, J. Biol. Chem./278(35):32861; MG132 (Calbiochem, La Jolla, Calif.); tosyl-Phe-chloromethylketone (“TPCK”); (N-6-chloro-7-methoxy-9H-.beta.-carbolin-8-yl)-2-methylnicotinamide (ML120B, Wen et al., 2006, J. Pharm. Exp. Ther. 317:989-1001; Celastrol (Lee, et al., 2006, Biochem. Pharmacol. 72(10): 1311-1321); PG201 (Shin, et al., 2005, Biochem. Biophys. Res. Common. 331(4): 1469-1477); and MLN0415 (Millennium Pharmaceuticals, Cambridge, Mass.); as being effective in blocking the interaction of IKK-α and -β. These compounds however were ineffective in vivo due to limited stability and biodistribution.